The anti-tumor effects of AZD4547 on ovarian cancer cells: differential responses based on c-Met and FGF19/FGFR4 expression.

BACKGROUND: The FGF/FGFR signaling pathway plays a critical role in human cancers. We analyzed the anti-tumor effect of AZD4547, an inhibitor targeting the FGF/FGFR pathway, in epithelial ovarian cancer (EOC) and strategies on overcoming AZD4547 resistance. METHODS: The effect of AZD4547 on cell viability/migration was evaluated and in vivo experiments in intraperitoneal xenografts using EOC cells and a patient-derived xenograft (PDX) model were performed. The effect of the combination of AZD4547 with SU11274, a c-Met-specific inhibitor, FGF19-specific siRNA, or an FGFR4 inhibitor was evaluated by MTT assay. RESULTS: AZD4547 significantly decreased cell survival and migration in drug-sensitive EOC cells but not drug-resistant cells. AZD4547 significantly decreased tumor weight in xenograft models of drug-sensitive A2780 and SKOV3ip1 cells and in a PDX with drug sensitivity but not in models with drug-resistant A2780-CP20 and SKOV3-TR cells. Furthermore, c-Met expression was high in SKOV3-TR and HeyA8-MDR cells, and co-administration of SU11274 and AZD4547 synergistically induced cell death. In addition, expressions of FGF19 and FGFR4 were high in A2780-CP20 cells. Combining AZD4547 with FGF19 siRNA or with a selective FGFR4 inhibitor led to significantly reduced cell proliferation in A2780-CP20 cells. CONCLUSIONS: This study showed that AZD4547 has significant anti-cancer effects in drug-sensitive cells and PDX models but not in drug-resistant EOC cells. In drug-resistant cells, the expression level of c-Met or FGF19/FGFR4 may be a predictive biomarker for AZD4547 treatment response, and a combination strategy of drugs targeting c-Met or FGF19/FGFR4 together with AZD4547 may be an effective therapeutic strategy for EOC.

Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer.

OBJECTIVE: Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. METHODS: The human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with BNZ-111, and cell proliferation, apoptosis, and cell cycle were assessed. RESULTS: It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating ß-3 tubulin expression to suppress microtubule dynamics. CONCLUSION: BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology.

Upregulated TGF-ß1 contributes to hyperglycaemia in type 2 diabetes by potentiating glucagon signalling.

AIMS/HYPOTHESIS: Glucagon-stimulated hepatic gluconeogenesis contributes to endogenous glucose production during fasting. Recent studies suggest that TGF-ß is able to promote hepatic gluconeogenesis in mice. However, the physiological relevance of serum TGF-ß levels to human glucose metabolism and the mechanism by which TGF-ß enhances gluconeogenesis remain largely unknown. As enhanced gluconeogenesis is a signature feature of type 2 diabetes, elucidating the molecular mechanisms underlying TGF-ß-promoted hepatic gluconeogenesis would allow us to better understand the process of normal glucose production and the pathophysiology of this process in type 2 diabetes. This study aimed to investigate the contribution of upregulated TGF-ß1 in human type 2 diabetes and the molecular mechanism underlying the action of TGF-ß1 in glucose metabolism. METHODS: Serum levels of TGF-ß1 were measured by ELISA in 74 control participants with normal glucose tolerance and 75 participants with type 2 diabetes. Human liver tissue was collected from participants without obesity and with or without type 2 diabetes for the measurement of TGF-ß1 and glucagon signalling. To investigate the role of Smad3, a key signalling molecule downstream of the TGF-ß1 receptor, in mediating the effect of TGF-ß1 on glucagon signalling, we generated Smad3 knockout mice. Glucose levels in Smad3 knockout mice were measured during prolonged fasting and a glucagon tolerance test. Mouse primary hepatocytes were isolated from Smad3 knockout and wild-type (WT) mice to investigate the underlying molecular mechanisms. Smad3 phosphorylation was detected by western blotting, levels of cAMP were detected by ELISA and levels of protein kinase A (PKA)/cAMP response element-binding protein (CREB) phosphorylation were detected by western blotting. The dissociation of PKA subunits was measured by immunoprecipitation. RESULTS: We observed higher levels of serum TGF-ß1 in participants without obesity and with type 2 diabetes than in healthy control participants, which was positively correlated with HbA1c and fasting blood glucose levels. In addition, hyperactivation of the CREB and Smad3 signalling pathways was observed in the liver of participants with type 2 diabetes. Treating WT mouse primary hepatocytes with TGF-ß1 greatly potentiated glucagon-stimulated PKA/CREB phosphorylation and hepatic gluconeogenesis. Mechanistically, TGF-ß1 treatment induced the binding of Smad3 to the regulatory subunit of PKA (PKA-R), which prevented the association of PKA-R with the catalytic subunit of PKA (PKA-C) and led to the potentiation of glucagon-stimulated PKA signalling and gluconeogenesis. CONCLUSIONS/INTERPRETATION: The hepatic TGF-ß1/Smad3 pathway sensitises the effect of glucagon/PKA signalling on gluconeogenesis and synergistically promotes hepatic glucose production. Reducing serum levels of TGF-ß1 and/or preventing hyperactivation of TGF-ß1 signalling could be a novel approach for alleviating hyperglycaemia in type 2 diabetes.

Combination effect of poly (ADP-ribose) polymerase inhibitor and DNA demethylating agents for treatment of epithelial ovarian cancer.

OBJECTIVE: Poly (ADP)-ribose polymerase inhibitors (PARPi) are effective clinical agents for treatment of epithelial ovarian cancer (EOC) harboring BRCA mutations as well as those without BRCA mutations. In this study, we evaluate the efficacy of combined PARPi and DNA methyltransferase inhibitor (DNMTi) in EOCs. METHODS: Expression levels of DNMT1 and PARP1 proteins in EOC cells were assessed using western blot analysis and immunohistochemistry. To evaluate the effects of co-treatment of PARPi (olaparib) and DNMTi (5-azacitidine, 5-AZA), we performed cell proliferation, apoptosis, and wound-healing assays in EOC cells. In addition, we performed in vivo experiments using both cell-line and patient-derived xenograft (PDX) models of EOC. RESULTS: The combination of olaparib and 5-AZA significantly inhibited cell proliferation and migration and induced apoptosis compared with olaparib or 5-AZA alone in EOC cell lines including A2780, HeyA8, A2780-CP20, and HeyA8-MDR. Moreover, in vivo experiments with this combination showed significantly decreased weight and nodule numbers of tumors in cell-line xenograft models with A2780 cells and a PDX model compared with control, olaparib, and 5-AZA groups. As a potential mechanism, the expression of intracellular reactive oxygen species (ROS) and its related proteins, including p-ERK, NRF2, p-p38, HO-1, and γH2AX, was affected in EOC cells. CONCLUSIONS: Co-treatment with PARPi and DNMTi had a significant anti-tumor effect in EOC cells. This combination might be a potential therapeutic strategy for EOCs.

Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer.

OBJECTIVE: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). METHODS: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. RESULTS: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P = 0.035 and P = 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P = 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis. CONCLUSION: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant.

Alternative splicing variant of the scaffold protein APPL1 suppresses hepatic adiponectin signaling and function.

Adiponectin is an adipocyte-derived hormone with antidiabetic activities that include increasing the sensitivity of cells to insulin. Adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif (APPL1) stimulates adiponectin signaling and promotes adiponectin's insulin-sensitizing effects by binding to two adiponectin receptors, AdipoR1 and AdipoR2, and the insulin receptor. In this study, we report an alternative splicing variant of APPL1 (APPL1sv) that is highly expressed in mouse liver, pancreas, and spleen tissues. The expression levels of APPL1sv in liver tissues were enhanced in a mouse model of obesity and diabetic dyslipidemia (i.e. db/db mice) and reduced in calorie-restricted mice compared with ad libitum-fed mice. APPL1sv overexpression or suppression inhibited or enhanced, respectively, adiponectin-stimulated phosphorylation of AMP protein kinase (AMPK) in mouse hepatocytes. We also found that APPL1sv binds to AdipoR1 and AdipoR2 under basal conditions and that adiponectin treatment reduces this binding. Overexpression of APPL1sv blocked adiponectin-induced interactions of APPL1 with the adiponectin receptors. Moreover, adenovirus-mediated and short hairpin RNA-based suppression of APPL1sv greatly reduced high fat diet-induced insulin resistance and hepatic glucose production in mice. Our study identifies a key suppressor of hepatic adiponectin signaling and insulin sensitivity, a finding that may shed light on identifying effective therapeutic targets for treating insulin resistance and type 2 diabetes.

Sphingosine kinase 1 as a potential therapeutic target in epithelial ovarian cancer.

Sphingosine kinase 1 (SK1) is over-expressed in multiple types of human cancer. SK1 has growth-promoting effects and has been proposed as a potential therapeutic target. We investigated the therapeutic effects of SK1 inhibition in epithelial ovarian carcinoma (EOC). SK1 siRNA or inhibitors were tested in EOC cell lines, including A2780, SKOV3ip1, A2780-CP20, SKOV3-TR, ES2 and RMG2. Cells were treated with SK inhibitor or FTY720, and cell proliferation, apoptosis, angiogenesis and invasion were examined by MTT, FACS, ELISA and wound-healing assays, respectively. In vivo experiments were performed to test the effects of FTY720 on tumor growth in orthotopic mouse xenografts of EOC cell lines A2780 or SKOV3ip1 and a patient-derived xenograft (PDX) model of clear cell ovarian carcinoma (CCC). Blocking SK1 with siRNA or inhibitors significantly reduced proliferation, angiogenesis and invasion, and increased apoptosis in chemosensitive (A2780 and SKOV3ip1) and chemoresistant (A2780-CP20, SKOV3-TR, ES2 and RMG2) EOC cells. SK1 inhibitors also decreased the intracellular enzymatic activity of SK1. Furthermore, FTY720 treatment significantly decreased the in vivo tumor weight in xenograft models of established cell lines (A2780 and SKOV3ip1) and a PDX model for CCC compared to control (p < 0.05). These results support therapeutic targeting of SK1 as a potential new strategy for EOC.

Lack of adiponectin leads to increased lymphocyte activation and increased disease severity in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-ß expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment.

Deficiency of APPL1 in mice impairs glucose-stimulated insulin secretion through inhibition of pancreatic beta cell mitochondrial function.

AIMS/HYPOTHESIS: Adaptor protein, phosphotyrosine interaction, pleckstrin homology domain and leucine zipper containing 1 (APPL1) is an adapter protein that positively mediates adiponectin signalling. Deficiency of APPL1 in the target tissues of insulin induces insulin resistance. We therefore aimed, in the present study, to determine its role in regulating pancreatic beta cell function. METHODS: A hyperglycaemic clamp test was performed to determine insulin secretion in APPL1 knockout (KO) mice. Glucose- and adiponectin-induced insulin release was measured in islets from APPL1 KO mice or INS-1(832/13) cells with either APPL1 knockdown or overproduction. RT-PCR and western blotting were conducted to analyse gene expression and protein abundance. Oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential were assayed to evaluate mitochondrial function. RESULTS: APPL1 is highly expressed in pancreatic islets, but its levels are decreased in mice fed a high-fat diet and db/db mice compared with controls. Deletion of the Appl1 gene leads to impairment of both the first and second phases of insulin secretion during hyperglycaemic clamp tests. In addition, glucose-stimulated insulin secretion (GSIS) is significantly decreased in islets from APPL1 KO mice. Conversely, overproduction of APPL1 leads to an increase in GSIS in beta cells. In addition, expression levels of several genes involved in insulin production, mitochondrial biogenesis and mitochondrial OCR, ATP production and mitochondrial membrane potential are reduced significantly in APPL1-knockdown beta cells. Moreover, suppression or overexproduction of APPL1 inhibits or stimulates adiponectin-potentiated GSIS in beta cells, respectively. CONCLUSIONS/INTERPRETATION: Our study demonstrates the roles of APPL1 in regulating GSIS and mitochondrial function in pancreatic beta cells, which implicates APPL1 as a therapeutic target in the treatment of type 2 diabetes.

Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator and is used for the treatment of uterine leiomyoma (a benign tumor). Uterine sarcoma which is highly malignant cancer with a poor prognosis is clinically resembled with uterine leiomyoma. There has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models. Cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1). Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. An in vivo efficacy of UPA was examined with uterine sarcoma cell line- and patient-derived xenograft (PDX) mice models. UPA inhibited cell growth in uterine sarcoma cell lines and primary culture cells from a PDX mouse (PDX-C). cDNA microarray analysis revealed that CCL2 was highly down-regulated by UPA. Phosphorylation and the total expression of STAT3 were inhibited by UPA. UPA also inhibited CCL2 and STAT3 in PDX-C. The inhibitory effect of UPA had not changed in the overexpression of PR and treatment of progesterone. In vivo efficacy studies with cell line-derived xenografts and a PDX model with leiomyosarcoma, a typical uterine sarcoma, demonstrated that UPA significantly decreased tumor growth. UPA had significant anti-tumor effects in uterine sarcoma through the inhibition of STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease.

Anti-cancer effect of fenbendazole-incorporated PLGA nanoparticles in ovarian cancer.

OBJECTIVE: Fenbendazole (FZ) has potential anti-cancer effects, but its poor water solubility limits its use for cancer therapy. In this study, we investigated the anti-cancer effect of FZ with different drug delivery methods on epithelial ovarian cancer (EOC) in both in vitro and in vivo models. METHODS: EOC cell lines were treated with FZ and cell proliferation was assessed. The effect of FZ on tumor growth in cell line xenograft mouse model of EOC was examined according to the delivery route, including oral and intraperitoneal administration. To improve the systemic delivery of FZ by converting fat-soluble drugs to hydrophilic, we prepared FZ-encapsulated poly(D,L-lactide-co-glycolide) acid (PLGA) nanoparticles (FZ-PLGA-NPs). We investigated the preclinical efficacy of FZ-PLGA-NPs by analyzing cell proliferation, apoptosis, and in vivo models including cell lines and patient-derived xenograft (PDX) of EOC. RESULTS: FZ significantly decreased cell proliferation of both chemosensitive and chemoresistant EOC cells. However, in cell line xenograft mouse models, there was no effect of oral FZ treatment on tumor reduction. When administered intraperitoneally, FZ was not absorbed but aggregated in the intraperitoneal space. We synthesized FZ-PLGA-NPs to obtain water solubility and enhance drug absorption. FZ-PLGA-NPs significantly decreased cell proliferation in EOC cell lines. Intravenous injection of FZ-PLGA-NP in xenograft mouse models with HeyA8 and HeyA8-MDR significantly reduced tumor weight compared to the control group. FZ-PLGA-NPs showed anti-cancer effects in PDX model as well. CONCLUSION: FZ-incorporated PLGA nanoparticles exerted significant anti-cancer effects in EOC cells and xenograft models including PDX. These results warrant further investigation in clinical trials.

Anticancer Activity of the Combination of Cabozantinib and Temozolomide in Uterine Sarcoma.

PURPOSE: To evaluate the anticancer effects of cabozantinib, temozolomide, and their combination in uterine sarcoma cell lines and mouse xenograft models. EXPERIMENTAL DESIGN: Human uterine sarcoma cell lines (SK-LMS-1, SK-UT-1, MES-SA, and SKN) were used to evaluate the anticancer activity of cabozantinib, temozolomide, and their combination. The optimal dose of each drug was determined by MTT assay. Cell proliferation and apoptosis were assessed 48 and 72 hours after the drug treatments. The tumor weights were measured in an SK-LMS-1 xenograft mouse model and a patient-derived xenograft (PDX) model of leiomyosarcoma treated with cabozantinib, temozolomide, or both. RESULTS: Given individually, cabozantinib and temozolomide each significantly decreased the growth and viability of cells. This inhibitory effect was more pronounced when cabozantinib (0.50 µmol/L) and temozolomide (0.25 or 0.50 mmol/L) were co-administered (P < 0.05). The combination of the drugs also significantly increased apoptosis in all cells. Moreover, this effect was consistently observed in patient-derived leiomyosarcoma cells. In vivo studies with SK-LMS-1 cell xenografts and the PDX model with leiomyosarcoma demonstrated that combined treatment with cabozantinib (5 mg/kg/d, per os administration) and temozolomide (5 mg/kg/d, per os administration) synergistically decreased tumor growth (both P < 0.05). CONCLUSIONS: The addition of cabozantinib to temozolomide offers synergistic anticancer effects in uterine sarcoma cell lines and xenograft mouse models, including PDX. These results warrant further investigation in a clinical trial.

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells.

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in colorectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data collectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels. [BMB Reports 2022; 55(4): 198-203].

Adiponectin Alleviates Diet-Induced Inflammation in the Liver by Suppressing MCP-1 Expression and Macrophage Infiltration.

Adiponectin is an adipokine that exerts insulin-sensitizing and anti-inflammatory roles in insulin target tissues including liver. While the insulin-sensitizing function of adiponectin has been extensively investigated, the precise mechanism by which adiponectin alleviates diet-induced hepatic inflammation remains elusive. Here, we report that hepatocyte-specific knockout (KO) of the adaptor protein APPL2 enhanced adiponectin sensitivity and prevented mice from developing high-fat diet-induced inflammation, insulin resistance, and glucose intolerance, although it caused fatty liver. The improved anti-inflammatory and insulin-sensitizing effects in the APPL2 hepatocyte-specific KO mice were largely reversed by knocking out adiponectin. Mechanistically, hepatocyte APPL2 deficiency enhances adiponectin signaling in the liver, which blocks TNF-α-stimulated MCP-1 expression via inhibiting the mTORC1 signaling pathway, leading to reduced macrophage infiltration and thus reduced inflammation in the liver. With results taken together, our study uncovers a mechanism underlying the anti-inflammatory role of adiponectin in the liver and reveals the hepatic APPL2-mTORC1-MCP-1 axis as a potential target for treating overnutrition-induced inflammation in the liver.

LRG1 is an adipokine that mediates obesity-induced hepatosteatosis and insulin resistance.

Dysregulation in adipokine biosynthesis and function contributes to obesity-induced metabolic diseases. However, the identities and functions of many of the obesity-induced secretory molecules remain unknown. Here, we report the identification of leucine-rich alpha-2-glycoprotein 1 (LRG1) as an obesity-associated adipokine that exacerbates high fat diet-induced hepatosteatosis and insulin resistance. Serum levels of LRG1 were markedly elevated in obese humans and mice compared with their respective controls. LRG1 deficiency in mice greatly alleviated diet-induced hepatosteatosis, obesity, and insulin resistance. Mechanistically, LRG1 bound with high selectivity to the liver and promoted hepatosteatosis by increasing de novo lipogenesis and suppressing fatty acid ß-oxidation. LRG1 also inhibited hepatic insulin signaling by downregulating insulin receptor substrates 1 and 2. Our study identified LRG1 as a key molecule that mediates the crosstalk between adipocytes and hepatocytes in diet-induced hepatosteatosis and insulin resistance. Suppressing LRG1 expression and function may be a promising strategy for the treatment of obesity-related metabolic diseases.

Proteomic analysis of osteoclast lipid rafts: the role of the integrity of lipid rafts on V-ATPase activity in osteoclasts.

Lipid rafts are membrane platforms for signaling molecules that regulate various cellular functions. During osteoclastogenesis, lipid rafts also have been shown to play a crucial role for cell fusion upon RANK/RANKL interaction and further activation of osteoclasts, resulting in bone resorption. To investigate proteins that localize in lipid rafts, we conducted two-dimensional (2D) gel electrophoresis followed by MALDI-TOF mass spectrometry. We identified 12 functional proteins among 34 proteins that were detectable by silver staining of the 2D gel. Among them, a subunit of vacuolar H(+)-ATPase that is involved in resorption activity of osteoclasts was identified showing an approximate molecular weight of 56.94 kDa and pI of 5.4. The solubilization of osteoclast membrane proteins followed by sucrose density gradient fractionation confirmed that V-ATPase colocalized with flotillin-1, a marker of lipid rafts, in low-density detergent-insoluble fractions. Disruption of rafts with cholesterol-sequestering agents or introduction of a dominant-negative mutant of TRAF6 impaired V-ATPase activity. These data indicate that integrity of lipid rafts regulates the activity of V-ATPase in osteoclasts, suggesting that cholesterol-lowering agents might be useful for inhibiting osteoclast-dependent bone resorption.

Anti-Cancer Activity of As4O6 and its Efficacy in a Series of Patient-Derived Xenografts for Human Cervical Cancer.

PURPOSE: To investigate the anti-cancer effects of tetraarsenic hexoxide (TAO, As4O6) in cervical cancer cell lines and in a series of patient-derived xenograft (PDX) mouse models. METHODS: Human cervical cancer cell lines, including HeLa, SiHa and CaSki, and human umbilical vein endothelial cells (HUVECs), were used to evaluate the anti-cancer activity of TAO. Cellular proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA) for matrix metallopeptidase 2 (MMP-2) and 9 (MMP-9) were assessed. The tumor weights of the PDXs that were given TAO were measured. The PDXs included primary squamous cell carcinoma, primary adenocarcinoma, recurrent squamous cell carcinoma, and recurrent adenocarcinoma. RESULTS: TAO significantly decreased cellular proliferation and increased apoptosis in cervical cancer cell lines and HUVEC. The functional studies on the cytotoxicity of TAO revealed that it inhibited the activation of Akt and vascular endothelial growth factor receptor 2 (VEGFR2). It also decreased the concentrations of MMP-2 in both cervical cancer cell lines and HUVECs. Active caspase-3 and p62 were both increased by the treatment of TAO, indicating increased rates of apoptosis and decreased rates of autophagy, respectively. In vivo studies with PDXs revealed that TAO significantly decreased tumor weight for both primary squamous cell carcinoma and adenocarcinoma of the cervix. However, this anti-cancer effect was not seen in PDXs with recurrent cancers. Nevertheless, the combination of TAO with cisplatin significantly decreased tumor weight in PDX models for both primary and recurrent cancers. CONCLUSIONS: TAO exerted inhibitory effects on angiogenesis, cellular migration, and autophagy, and it showed stimulatory effects on apoptosis. Overall, it demonstrated anti-cancer effects in animal models for human cervical cancer.

Chloroquine reverses chemoresistance via upregulation of p21WAF1/CIP1 and autophagy inhibition in ovarian cancer.

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.